This paper adds new insight to a molecular phylogeny of Gyrodactylus, based on a complete sequence of the ITS rDNA region of 4 subgenera and a more detailed molecular analysis. We propose a hierarchical approach in elucidating the phylogeny of this species-rich genus. A total of 37 sequences (915–1239 bp) from 10 representative species from 4 out of 6 subgenera, as defined by Malmberg (1970), are included in the analysis. Genetic differences observed at the 5.8S locus provide objective criteria to separate (sub)genera, while deep genetic differences of the spacers form a sound basis for species-specific identification. We demonstrate that each Gyrodactylus subgenus possesses a unique sequence of the 5.8S gene. Thus, there is concordance between the 5.8S gene and the excretory system used by Malmberg (1970) as a diagnostic character of subgenus status. At the species level, there is a discrepancy between morphological and molecular variation. Whereas the morphological variation, expressed in the shape and size of the attachment apparatus, is very low, the molecular variation, expressed at the ITS1 and ITS2 regions, is very high. This can be attributed either to the fast evolving ITS region or to the fact that the genus consists of groups of a higher taxonomic level than previously recognized. Perspectives and limitations of using the ITS rDNA region for a molecular phylogeny of this genus are discussed.